ABSTRACT Patients with advanced breast cancer have a high propensity to metastasize to the brain with human epidermal growth factor receptor (EGFR) positive and triple-negative breast cancer (TNBC; estrogen receptor, progesterone receptor and Her2 negative) subtypes showing the highest incidence of brain metastases. Most patients have multiple metastatic lesions at the time of diagnosis making surgery an inadequate therapeutic option on its own. Furthermore, impaired cognitive decline induced by whole-brain radiation therapy (WBRT) and the tight blood brain barrier (BBB) preventing the brain permeability of systemic therapies in the brain pose challenges for the success of existing therapies and result in failure to improve overall patient survival. To effectively treat multiple highly aggressive breast metastatic foci in the brain, there is an urgent need to develop tumor specific multi-targeting agents that simultaneously target multiple aberrant signaling pathways in TNBC and utilize delivery vehicles which specifically seek metastatic foci in the brain. In our previously published and preliminary studies, we have 1) extensively demonstrated that engineered human and mouse neural stem cells (NSC) and mesenchymal stem cells (MSC) home extensively to primary and metastatic tumors in the brain and provide on-site means to deliver novel tumor specific agents; and 2) engineered EGFR-specific nanobodies (ENb) and their pro-apoptotic variant, bi- functional ENb-TRAIL and shown its potential to target both cell proliferation and death pathways in a mechanism based manner in broad spectrum of tumor cells. The long term goal of this proposal is to test the mechanism based therapeutic efficacy of systemically delivered NSC-ENb-TRAIL in TNBC derived mouse models of breast to brain metastasis that mimic the clinical scenario of breast metastatic tumor growth and progression. We will initially screen established and patient derived TNBC lines for their response to EGFR and DR4/5 targeted therapies and assess their propensity to metastasize to brain. The mechanism based response of TNBC to NSC-ENb-TRAIL and the fate and therapeutic efficacy of NSC-ENb-TRAIL in brain metastasis mouse models generated from brain seeking patient derived TNBC lines will be assessed. We hypothesize that simultaneous targeting of EGFR and DR4/5 will significantly influence epithelial-mesenchymal transition (EMT) and tumor growth and progression. Based on our exciting preliminary data on the combined use BBB permeable histone deacetylase inhibitor (HDACi), CN147 and ENb-TRAIL and the previous findings that concomitant use of HDACi with DR4/5 agonists and EGFR inhibitors, the combined therapeutic efficacy of NSC-ENb-TRAIL and CN147 will be assessed. We hypothesize that ENb- TRAIL and HDACi will have therapeutic efficacy in metastatic TNBC with a broader range of genetic backgrounds and with varying sensitivity to ENb-TRAIL in vivo. The proposed studies in this application are likely to unravel the mechanism-based, targeted stem cell mediated therapies for metastatic breast tumors. We envision designing a strategy in which NSC-ENb-TRAIL will be injected intra-arterially to target the metastatic tumor deposits in the brain of metastatic breast cancer patients. This will have a major impact in saving the lives of many cancer patients.